Lung surfactant protein B promoter function is dependent on the helical phasing, orientation and combinatorial actions of cis-DNA elements
Introduction
Surfactant, a complex of lipids and proteins, is synthesized and secreted by the type II epithelial cells of the pulmonary alveolus. Surfactant maintains the integrity of the alveoli during respiration by reducing surface tension at the alveolar air-tissue interface (Goerke and Clements, 1986) and serves important roles in host defense in the lung (Van Iwaarden and Van Golde, 1995). Deficiency of surfactant is directly linked to the development of respiratory distress syndrome (RDS) in premature infants (Avery and Mead, 1959), the leading cause of neonatal morbidity and mortality in developed countries. Surfactant levels are also reduced in adult respiratory distress syndrome (Lewis and Jobe, 1993), a disease with a high mortality rate. Surfactant protein B (SP-B), a 9 kDa hydrophobic protein of surfactant, is essential for the maintenance of biophysical properties and physiological function of surfactant. Studies have shown that SP-B promotes the adsorption and spreading of phospholipids (Possmayer, 1990) and stabilizes the phospholipid monolayer formed on the alveolar surface (Cochrane and Revak, 1991). The critical role of SP-B in surfactant function is underscored by its deficiency in congenital alveolar proteinosis in newborns (Nogee et al., 1993). Infants with congenital alveolar proteinosis develop respiratory failure at birth and succumb to death without lung transplantation. Targeted disruption of SP-B gene caused respiratory failure in newborn mice, further supporting the important role of SP-B in lung function (Clark et al., 1995).
SP-B gene is expressed in a cell/tissue-specific manner by the alveolar type II and bronchiolar (Clara) epithelial cells of the lung (Phelps and Floros, 1988, Wohlford-Lenane and Snyder, 1992) and is developmentally regulated. SP-B gene expression is also regulated in a multifactorial manner by hormones, cytokines and growth factors (Boggaram, 2000). Our previous studies showed that a minimal promoter region containing −236/+39 bp of rabbit SP-B gene is necessary and sufficient for optimal promoter activity in H441 cells (Margana and Boggaram, 1996), a cell line of bronchiolar epithelial or Clara cell lineage and in MLE-12 cells (Adams et al., 2001), a cell line with characteristics of alveolar type II epithelial cells. The rabbit SP-B minimal promoter contained functionally important cis-DNA elements for binding of Sp1/Sp3, thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3 (HNF-3), activating transcription factor/cAMP response element binding protein (ATF/CREB) transcription factors (Margana and Boggaram, 1997, Margana et al., 2000, Berhane and Boggaram, 2001). Further our studies on SP-B promoter regulation in transgenic mice identified SP-B genomic region −730/+39 bp to contain cis-DNA elements necessary for lung cell-specific and developmental expression of chloramphenicol acetyltransferase (CAT) reporter gene (Adams et al., 2001). The SP-B minimal promoter, −236/+39 bp, although maintained lung-cell specific expression did not express CAT to the same level as the −730/+39 bp region in transgenic mice (Adams et al., 2001).
The minimal promoter regions of human (Pilot-Matias et al., 1989, Bohinski et al., 1994), rabbit (Margana and Boggaram, 1996) and mouse (Bruno et al., 1995) SP-B genes display a high degree of sequence similarity and contain cis-DNA elements for the binding of Sp1/Sp3, TTF-1, HNF-3 and ATF/CREB transcription factors. In addition to significant conservation of nucleotide sequence, the spacing and the orientation of cis-DNA elements in the minimal promoter regions of human, rabbit and mouse SP-B genes are very similar (Margana and Boggaram, 1996). Based on these data we hypothesized that orientation and helical phasing/stereospecific alignment between the cis-DNA elements play important roles in promoter activation.
In the present investigation we studied the importance of the roles of helical phasing and orientation of cis-DNA elements on SP-B promoter function in H441 cells. Additionally we studied the effects of transcription factors on SP-B promoter function by cotransfection experiments. To address these questions, we altered: (1) the distance between the cis-DNA elements by introducing half-helical (5 bp) and full-helical (10 bp) turns of DNA; and (2) orientation of cis-DNA elements, and assessed their effects on SP-B promoter activity by transient transfection analysis. We found that insertion of half-helical turn of DNA between HNF-3 and Sp1 (−35 bp) sites or between the enhancer and the TATA element significantly reduced the promoter activity whereas insertion of a full-helical turn partially reversed the inhibitory effects of half-helical turn of DNA. Inversion of orientation of HNF-3 and Sp1 (−35 bp) binding sites compared to the wild type promoter caused significant reduction of SP-B promoter activity. These data suggested that proper helical phasing and orientation of cis-DNA elements are necessary for SP-B promoter function. Co-transfection experiments showed that transcription factors act in a combinatorial rather than in a synergistic manner to enhance SP-B promoter activity.
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Cell culture and transfections
NCI-H441 (ATCC HTB-174), a human lung adenocarcinoma cell line with characteristics of bronchiolar or Clara epithelial cells and A549 (ATCC CCL-185), a human lung carcinoma epithelial cell line were maintained in RPMI 1640 and F-12K media, respectively, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B at 37°C in a humidified atmosphere of 5% CO2 and air.
Plasmid DNAs were purified by anion exchange chromatography using a kit
Effects of alterations in helical phasing between the cis-DNA elements on SP-B promoter activity
The minimal promoter sequences of rabbit, human and mouse SP-B genes are similar indicating a high degree of sequence conservation (Margana and Boggaram, 1996). In the rabbit SP-B promoter, functionally important cis-DNA elements for Sp1, TTF-1 and HNF-3 are clustered over a region of 70 nucleotides (Margana and Boggaram, 1997). The locations, spacing between the DNA elements and orientations of these cis-DNA elements in the minimal promoters of rabbit, human and mouse SP-B genes are similar (
Helical phasing and orientation of cis-DNA elements are important for SP-B promoter function
The developmental induction and lung cell/tissue-specific expression of SP-B mRNA are tightly regulated. The proximal promoters of rabbit, human and mouse SP-B genes display significant conservation with regard to nucleotide sequence as well as spacing and orientation of cis-DNA elements. The strict conservation of spacing and orientation of the DNA elements suggested that assembly of a stereospecific transcription complex may be necessary for SP-B promoter function. Although cis-DNA elements
Acknowledgements
This work was supported by the National Institutes of Health Grant HL-48048. We acknowledge Dr Ramgopal Margana for his contributions and thank Mrs Ismat Alam for excellent technical assistance.
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